Erythropoietin (Epo) regulates the production of red blood cells by binding to its cell surface receptor EpoR expressed on erythroid progenitor cells. The biologic effects of Epo are not limited to the regulation of mammalian erythropoiesis. In addition to impaired fetal liver erythropoiesis, Epo or EpoR-null mouse embryos exhibit defects in cardiac morphogenesis, abnormalities in the vascular network and increased apoptosis in endocardium and myocardium. Although Epo-EpoR function has been implicated in normal cardiac development, little is known about direct physiologic effects of Epo-EpoR signaling in the heart. We investigated cardiac effects of Epo and tested the hypothesis that Epo may exert a cardioprotective effect during global cardiac ischemia followed by reperfusion. We found that Epo exhibits a positive inotropic and cardioprotective effect with significantly improved post-ischemia contractile recovery following ischemiareperfusion injury in an experimental model of isolated, perfused hearts. The objective of this proposal is to investigate Epo-EpoR signaling in the heart with focus on the mechanisms involved in the ability of Epo to protect cardiomyocytes from ischemic damage. The specific aims of this proposal are 1). To examine the effect of Epo on infarct size and apoptosis during cardiac ischemia-reperfusion to test the hypothesis that Epo may reduce infarct size and cardiomyocyte apoptosis in an experimental model of isolated rat heart perfusion. 2). To characterize Epo-EpoR signal transduction pathways in cultured primary cardiomyocytes and in ex vivo perfused hearts to test the hypothesis that Epo treatment of cardiomyocytes may result in activation of functional downstream EpoR signaling pathways including phosphatidylinositol 3-kinase (PI3-K) and protein kinase B/Akt pathway. 3). To investigate signaling mechanisms involved in Epo-mediated cardioprotective effect during ischemia-reperfusion to test the hypothesis that the cardioprotective effects of Epo may be associated with EpoR-mediated activation of PI3-kinase/Akt pathway. These studies will begin to characterize functional EpoR signaling pathways in the heart, investigate the mechanisms of the cardioprotective effects of Epo and lay the groundwork for future clinical trials evaluating the use of recombinant human Epo as cardioprotective therapy, in syndromes of acute ischemic cardiac injury.